Human ß-Defensin 23 as a Carrier for In Vitro and In Vivo Delivery of mRNA.

The successful application of mRNA therapeutics hinges on the effective intracellular delivery of mRNA both in vitro and in vivo. However, this remains a formidable challenge due to the polyanionic nature, longitudinal shape, and low nuclease resistance of mRNA. In this study, we introduce a novel mRNA delivery platform utilizing a human ß-defensin peptide, hBD23. The positive charge of hBD23 allows it to form nanocomplexes with mRNA, facilitating cellular uptake and providing protection against serum nucleases. When optimized for peptide-to-mRNA (N/P) ratios, these hBD23/mRNA complexes demonstrated efficient cellular delivery and subsequent protein expression both in vitro and in vivo. Importantly, as hBD23 is human derived, the complexes exhibited minimal cytotoxicity and immunogenicity. Given its high biocompatibility and delivery efficiency, hBD23 represents a promising platform for the in vitro and in vivo delivery of mRNA.

Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability.

Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).

Cholesterol-Mediated Seeding of Protein Corona on DNA Nanostructures for Targeted Delivery of Oligonucleotide Therapeutics to Treat Liver Fibrosis.

The protein corona is a protein layer formed on the surface of nanoparticles administered in vivo and considerably affects the in vivo fate of nanoparticles. Although it is challenging to control protein adsorption on nanoparticles precisely, the protein corona may be harnessed to develop a targeted drug delivery system if the nanoparticles are decorated with a ligand with enhanced affinity to target tissue- and cell-homing proteins. Here, we prepared a DNA tetrahedron with trivalent cholesterol conjugation (Chol3-Td) that can induce enhanced interaction with lipoproteins in serum, which in situ generates the lipoprotein-associated protein corona on a DNA nanostructure favorable for cells abundantly expressing lipoprotein receptors in the liver, such as hepatocytes in healthy mice and myofibroblasts in fibrotic mice. Chol3-Td was further adopted for liver delivery of antisense oligonucleotide (ASO) targeting TGF-ß1 mRNA to treat liver fibrosis in a mouse model. The potency of ASO@Chol3-Td was comparable to that of ASO conjugated with the clinically approved liver-targeting ligand, trivalent N-acetylgalactosamine (GalNAc3), demonstrating the potential of Chol3-Td as a targeted delivery system for oligonucleotide therapeutics. This study suggests that controlled seeding of the protein corona on nanomaterials can provide a way to steer nanoparticles into the target area.

Kissing loop-mediated fabrication of RNA nanoparticles and their potential as cellular and in vivo siRNA delivery platforms.

We describe an efficient method to condense RNAs into tightly packed RNA nanoparticles (RNPs) for biomedical applications without hydrophobic or cationic agents. We embedded kissing loops and siRNA in the RNAs to constrain the size of RNPs to ca. 100 nm, making them suitable not only for cellular uptake but also for passive tumor accumulation. The resulting RNPs were efficiently internalized into cells and downregulated the target gene of siRNAs. When intravenously injected into tumor-bearing mice, RNPs could also accumulate in the tumor. The reported fabrication method could be readily adopted as a platform to prepare RNPs for in vitro and in vivo delivery of bioactive RNAs.

Kidney-Targeted Cytosolic Delivery of siRNA Using a Small-Sized Mirror DNA Tetrahedron for Enhanced Potency.

A proper intracellular delivery method with target tissue specificity is critical to utilize the full potential of therapeutic molecules including siRNAs while minimizing their side effects. Herein, we prepare four small-sized DNA tetrahedrons (sTds) by self-assembly of different sugar backbone-modified oligonucleotides and screened them to develop a platform for kidney-targeted cytosolic delivery of siRNA. An in vivo biodistribution study revealed the kidney-specific accumulation of mirror DNA tetrahedron (L-sTd). Low opsonization of L-sTd in serum appeared to avoid liver clearance and keep its size small enough to be filtered through the glomerular basement membrane (GBM). After GBM filtration, L-sTd could be delivered into tubular cells by endocytosis. The kidney preference and the tubular cell uptake property of the mirror DNA nanostructure could be successfully harnessed for kidney-targeted intracellular delivery of p53 siRNA to treat acute kidney injury (AKI) in mice. Therefore, L-sTd could be a promising platform for kidney-targeted cytosolic delivery of siRNA to treat renal diseases.

Systemic delivery of aptamer-drug conjugates for cancer therapy using enzymatically generated self-assembled DNA nanoparticles.

Aptamer-drug conjugates (ApDCs) are promising anticancer therapeutics with cancer cell specificity. However, versatile in vivo applications of ApDCs are hampered by their limited serum stability and inability to reach the tumour upon systemic administration. Here, we describe DNA nanoparticles of ApDCs as a platform for tumour-targeted systemic delivery of ApDCs. DNA nanoparticles of approximately 75 nm size were fabricated by self-assembly of a polymerised floxuridine (FUdR)-incorporated AS1411 aptamer produced via rolling circle amplification. The DNA nanoparticles of ApDCs showed highly efficient cancer cell uptake, enhanced serum stability, and tumour-targeted accumulation. These properties could be successfully utilised for tumour-specific apoptotic damage by ApDCs, leading to significant suppression of tumour growth without considerable systemic toxicity. Molecular analysis revealed that the enhanced anticancer potency was due to the synergic effect induced by the simultaneous activation of p53 by AS1411 and the inhibition of thymidylate synthase by FUdR, respectively, both of which were generated from the DNA nanoparticles. We therefore expect that the DNA nanoparticles of ApDCs can be a promising platform for tumour-targeted delivery of various nucleoside-incorporated ApDCs to treat cancer.

A brain tumor-homing tetra-peptide delivers a nano-therapeutic for more effective treatment of a mouse model of glioblastoma.

Organ-specific cell-penetrating peptides (CPPs) are a class of molecules that can be highly effective at delivering therapeutic cargoes, and they are currently of great interest in cancer treatment strategies. Herein, we describe a new CPP (amino acid sequence serine-isoleucine-tyrosine-valine, or SIWV) that homes to glioblastoma multiforme (GBM) brain tumor tissues with remarkable specificity in vitro and in vivo. The SIWV sequence was identified from an isoform of annexin-A3 (AA3H), a membrane-interacting human protein. The mechanism of intracellular permeation is proposed to follow a caveolin-mediated endocytotic pathway, based on in vitro and in vivo receptor inhibition and genetic knockdown studies. Feasibility as a targeting agent for therapeutics is demonstrated in a GBM xenograft mouse model, where porous silicon nanoparticles (pSiNPs) containing the clinically relevant anticancer drug SN-38 are grafted with SIWV via a poly-(ethylene glycol) (PEG) linker. The formulation shows enhanced in vivo targeting ability relative to a formulation employing a scrambled control peptide, and significant (P < 0.05) therapeutic efficacy relative to free SN-38 in the GBM xenograft animal model.

Performance of a novel fluorogenic probe assay for the detection of extended-spectrum-ß-lactamase or plasmid AmpC ß-lactamase-producing Enterobacterales directly from simulated blood culture bottles.

Resistance to third generation cephalosporins is widely disseminated in Enterobacteriaceae mainly because of extended-spectrum-ß-lactamases (ESBL), plasmid AmpC ß-lactamases (PABL), and hyper-production of chromosomal AmpC ß-lactamases. Here, we evaluated the performance of rapid test using novel fluorogenic probe assay in simulated blood cultures and compared the results with the phenol red assay using a total of 172 characterized isolates (39 ESBL producers, 13 PABL producers, and 120 susceptible isolates). We prepared a pellet by centrifugation and washing, which can also be used for identification with MALDI-TOF directly from positive blood cultures. After that, we mixed the pellet with fluorogenic probe and measured the fluorescent signal using fluorometer. The fluorogenic probe assay showed higher sensitivity than the phenol red assay (96.2% vs. 71.2%, p < .0001) in 172 simulated blood culture bottles especially in detecting PABL (84.6% vs. 0%, p = .0026) and the turnaround time was 1.5 h. This fluorogenic probe assay, combined with the direct identification of pathogens, could be very useful for rapid identification of isolates and detecting cephalosporin resistance caused by ESBL and PABL directly from positive blood cultures.

Lung-targeted delivery of TGF-ß antisense oligonucleotides to treat pulmonary fibrosis.

Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-ß is a critical factor in the fibrotic process, downregulation of this cytokine has been considered a potential approach for disease treatment. Herein, we designed a new lung-targeted delivery technology based on the complexation of polymeric antisense oligonucleotides (pASO) and dimeric human ß-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-ß mRNA were polymerized by rolling circle amplification and complexed with DhBD23. After complexation with DhBD23, pASO showed improved serum stability and enhanced uptake by fibroblasts in vitro and lung-specific accumulation upon intravenous injection in vivo. The pASO/DhBD23 complex delivered into the lung downregulated target mRNA, and subsequently alleviated lung fibrosis in mice, as demonstrated by western blotting, quantitative reverse-transcriptase PCR (qRT-PCR), immunohistochemistry, and immunofluorescence imaging. Moreover, as the complex was prepared only with highly biocompatible materials such as DNA and human-derived peptides, no systemic toxicity was observed in major organs. Therefore, the pASO/DhBD23 complex is a promising gene therapy platform with lung-targeting ability to treat various pulmonary diseases, including pulmonary fibrosis, with low side effects.

The crystal structure of a natural DNA polymerase complexed with mirror DNA.

The intrinsic l-DNA binding properties of a natural DNA polymerase was discovered. The binding affinity of Dpo4 polymerase for l-DNA was comparable to that for d-DNA. The crystal structure of Dpo4/l-DNA complex revealed a dimer formed by the little finger domain that provides a binding site for l-DNA.

A self-assembled DNA tetrahedron as a carrier for in vivo liver-specific delivery of siRNA.

While siRNA is a potent therapeutic tool that can silence disease-causing mRNA, its in vivo potency can be compromised due to the lack of target tissue specificity. Here, we report a wireframe tetrahedral DNA nanostructure having a 20-mer duplex on each side that can be specifically distributed into the liver upon systemic administration. This liver-targeted DNA tetrahedron is employed as the carrier for liver-specific delivery of siRNA targeting ApoB1 mRNA, which is overexpressed in hypercholesterolemia. When delivered by a DNA tetrahedron, the siRNA can preferentially be accumulated in the liver and down-regulate the ApoB1 protein. As a result, the blood cholesterol level is also decreased by the siRNA. These results successfully demonstrate that the DNA tetrahedron is a promising carrier for liver-targeted delivery of therapeutic nucleic acids.

Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity.

We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.

Modulating α-synuclein fibril formation using DNA tetrahedron nanostructures.

The small presynaptic protein α-synuclein (α-syn) is involved in the etiology of Parkinson's disease owing to its abnormal misfolding. To date, little information is known on the role of DNA nanostructures in the formation of α-syn amyloid fibrils. Here, the effects of DNA tetrahedrons on the formation of α-syn amyloid fibrils were investigated using various biochemical and biophysical methods such as thioflavin T fluorescence assay, atomic force microscopy, light scattering, transmission electron microscopy, and cell-based cytotoxicity assay. It has been shown that DNA tetrahedrons decreased the level of oligomers and increased the level of amyloid fibrils, which corresponded to decreased cellular toxicity. The ability of DNA tetrahedron to facilitate the formation of α-syn amyloid fibrils demonstrated that structured nucleic acids such as DNA tetrahedrons could modulate the process of amyloid fibril formation. Our study suggests that DNA tetrahedrons could be used as an important facilitator toward amyloid fibril formation of α-synuclein, which may be of significance in finding therapeutic approaches to Parkinson's disease and related synucleinopathies.

Highly tumor-specific DNA nanostructures discovered by in vivo screening of a nucleic acid cage library and their applications in tumor-targeted drug delivery.

Enormous efforts have been made to harness nanoparticles showing extravasation around tumors for tumor-targeted drug carriers. Owing to the complexity of in vivo environments, however, it is very difficult to rationally design a nanoconstruct showing high tumor specificity. Here, we show an approach to develop tumor-specific drug carriers by screening a library of self-assembled nucleic acid cages in vivo. After preparation of a library of 16 nucleic acid cages by combining the sugar backbone and the shape of cages, we screened the biodistribution of the cages intravenously injected into tumor-bearing mice, to discover the cages with high tumor-specificity. This tumor specificity was found to be closely related with serum stability, cancer cell uptake efficiency, and macrophage evasion rate. We further utilized the cages showing high tumor specificity as carriers for the delivery of not only a cytotoxic small molecule drug but also a macromolecular apoptotic protein exclusively into the tumor tissue to induce tumor-specific damage. The results demonstrate that our library-based strategy to discover tumor-targeted carriers can be an efficient way to develop anti-cancer nanomedicines with tumor specificity and enhanced potency.

Shaping Rolling Circle Amplification Products into DNA Nanoparticles by Incorporation of Modified Nucleotides and Their Application to In Vitro and In Vivo Delivery of a Photosensitizer.

Rolling circle amplification (RCA) is a robust way to generate DNA constructs, which are promising materials for biomedical applications including drug delivery because of their high biocompatibility. To be employed as a drug delivery platform, however, the DNA materials produced by RCA need to be shaped into nanoparticles that display both high cellular uptake efficiency and nuclease resistance. Here, we showed that the DNA nanoparticles (DNPs) can be prepared with RCA and modified nucleotides that have side-chains appended on the nucleobase are capable of interacting with the DNA strands of the resulting RCA products. The incorporation of the modified nucleotides improved cellular uptake efficiency and nuclease resistance of the DNPs. We also demonstrated that these DNPs could be employed as carriers for the delivery of a photosensitizer into cancer cells to achieve photodynamic therapy upon irradiation at both the in vitro and in vivo levels.

Tetrahedral DNAzymes for enhanced intracellular gene-silencing activity.

We prepared tetrahedral DNAzymes (TDzs) to overcome potential limitations such as insufficient serum stability and poor cellular uptake of single-stranded DNAzymes (ssDzs). TDzs showed enhanced serum stability and higher cellular uptake efficiency compared to those of ssDzs, providing significantly improved intracellular gene-silencing activity to down-regulate the target mRNA level.

Streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular and tumor delivery of enzymes.

Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs.

Reversible Regulation of Enzyme Activity by pH-Responsive Encapsulation in DNA Nanocages.

Reversible regulation of enzyme activity by chemical and physical stimuli is often achieved by incorporating stimuli-responsive domains in the enzyme of interest. However, this method is suitable for a limited number of enzymes with well-defined structural and conformational changes. In this study, we present a method to encapsulate enzymes in a DNA cage that could transform its conformation depending on the pH, allowing reversible control of the accessibility of the enzyme to the surrounding environment. This enabled us to regulate various properties of the enzyme, such as its resistance to protease-dependent degradation, binding affinity to the corresponding antibody, and most importantly, enzyme activity. Considering that the size and pH responsiveness of the DNA cage can be easily adjusted by the DNA length and sequence, our method provides a broad-impact platform for controlling enzyme functions without modifying the enzyme of interest.

In vitro and in vivo behavior of DNA tetrahedrons as tumor-targeting nanocarriers for doxorubicin delivery.

Deoxyribonucleic acid (DNA) is a versatile material with high applicability and inherent biocompatibility. L-DNA, the perfect mirror form of the naturally occurring D-DNA, has been used in DNA nanotechnology. It has thermodynamically identical properties to D-DNA, is capable of self-assembly and bio-orthogonal base-pairing, and is resistant to nuclease activity. We previously constructed an L-DNA tetrahedron (L-Td) and found that this nanostructure has remarkably higher capacity for cell penetration than its natural counterpart (D-Td). L-Td molecules of two different sizes-one with 17-mer per side (L-Td17) and the other with 30-mer per side (L-Td30)-were prepared by assembling four L-DNA strands. In this study, cellular uptake of L-Td with different sizes was observed over time using a laser scanning confocal microscope (LSCM) equipped with a live cell chamber system. In addition, we conducted a pharmacokinetic study to examine the potential of L-Td as a carrier for in vivo tumor-targeted delivery of a low dose of doxorubicin (DOX). L-Td entered into the cells through endocytosis, and a specific DNA sequence of the L-Td ensures targeted entry into cancer cells. Compared with free DOX, DOX-loaded L-Td (DOX@L-Td) showed decreased clearance and increased initial concentration (C0), half-life, and area under the curve (AUC), indicating that DOX@L-Td circulated in the blood stream for longer than free DOX. L-Td17, in particular, had beneficial effects owing to its ability to enhance tumor accumulation of DOX and reduce the cardiotoxicity caused by it through administration of a low dose of the drug.

Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) can be used for the specific disruption of a target gene to permanently suppress the expression of the protein encoded by the target gene. Efficient delivery of the system to an intracellular target site should be achieved to utilize the tremendous potential of the genome-editing tool in biomedical applications such as the knock-out of disease-related genes and the correction of defect genes. Here, we devise polymeric CRISPR/Cas9 system based on poly-ribonucleoprotein (RNP) nanoparticles consisting of polymeric sgRNA, siRNA, and Cas9 endonuclease in order to improve the delivery efficiency. When delivered by cationic lipids, the RNP nanoparticles built with chimeric poly-sgRNA/siRNA sequences generate multiple sgRNA-Cas9 RNP complexes upon the Dicer-mediated digestion of the siRNA parts, leading to more efficient disruption of the target gene in cells and animal models, compared with the monomeric sgRNA-Cas9 RNP complex.